2.7. mtROS
The MitoSOX™ Red mitochondrial superoxide indicator (M36008; Invitrogen) was used to detect mitochondrial ROS (mtROS) accumulation. The cells were incubated with 1.0 mL of 5 μM MitoSOX™ reagent working solution for 10 min at 37°C in dark. Thereafter, the cells were washed gently with warm buffer thrice, and fluorescence was observed and photographed under a fluorescence microscope (DMI4000B; Leica, Wetzlar, Germany).